Introduction

Non–Hodgkin lymphomas (NHL) are a heterogeneous group of malignancies of the lymphoid system. Current treatments for NHL are not optimally effective as both relapse and resistance to common chemotherapy are usually observed (1–4). Monoclonal antibodies have revolutionized the treatment of malignances such as NHL.

Rituximab is an anti-CD20 monoclonal antibody that has been approved by the U.S. Food and Drug Administration for the treatment of CD20+ B-NHL (5, 6). It induces cell lysis through antibody-dependent cell-mediated cytotoxicity, complement-dependent cytotoxicity, and apoptosis (7, 8). There has not been an increase in overall survival (5, 6). Approximately 50% of treated patients are refractory or develop resistance during the course of prolonged treatment with rituximab as single agent or in combination with cyclophosphamide-Adriamycin-vincristine-prednisone (CHOP; ref. 9). Because resistance to rituximab occurs, there is a need for new therapies (10).

CD80 is a membrane-bound immune costimulatory molecule involved in the regulation of the activation of T cells (11). It is a member of the B7 family of costimulatory molecules (12). CD80 is expressed on the surface of normal activated B cells, antigen-presenting cells, and T cells (13 and on the surface of a variety of lymphoid malignances (14–17). Preclinical studies have showed that anti-CD80 antibodies can inhibit cell proliferation of lymphoma and induce antibody-dependent cell-mediated cytotoxicity (18) in vivo. Conjugated anti-CD80 antibodies with immunotoxins exerted antitumor activity in vitro using CD80+ Burkitt lymphoma (Raji) and Hodgkin lymphoma (L428) cell lines (19). Galiximab delays progression and prolongs survival in a human lymphoma xenograft/severe combined immunodeficient (SCID) mouse model (20). Previous findings showed that treatment with a monoclonal antibody for CD80 (16-10A1) on B cells resulted in a significant decrease in tumor cell proliferation, upregulation of proapoptotic molecules, and downregulation of the expression of antiapoptotic proteins such as Bcl-XL, thus, resulting in tumor cell apoptosis (21).

Galiximab serves as a counter receptor that transduces distinct signaling to B cells upon engagement. Several studies have reported the signaling on T cells through the CD80 ligand (CD28) via interaction with CD80-expressing cells (25). However, little is known about the direct signaling by galiximab of cells expressing CD80. These results indicated that CD80 can mediate signal transduction and regulate B-cell function (21).

We and others have reported that rituximab treatment affects apoptotic signaling in lymphoma cell lines via upstream inhibition of constitutively activated survival signaling pathways such as the p38MAPK and NF-κB (26, 27) and downstream inhibition of Bcl-2 and Bcl-XL expression and in the reversal of resistance (28). In addition, treatment of B-NHL cell lines with rituximab resulted in potentiation of apoptosis induced by Fas-L and TRAIL, respectively, through the inhibition of the transcriptional repressor YY1 (29, 30). Furthermore, NF-κB activates the transcription of resistant gene products such as Snail (31–33).

Galiximab interferes with the apoptotic pathways by targeting gene products regulated by NF-κB. Therefore, we hypothesized that galiximab may inhibit the constitutively activated NF-κB pathway and may also sensitize resistant B-NHL cell lines to apoptosis by cytotoxic drugs. The following were examined to test this hypothesis: (i) Does treatment of CD80+ Burkitt B-NHL cell lines inhibit the constitutively activated NF-κB and AKT pathways; (ii) Does treatment of B-NHL cell lines with galiximab sensitize the cells to apoptosis by chemotherapeutic and immunotherapeutic drugs?; and (iii) Does galiximab-mediated sensitization to apoptosis result from inhibition of resistant factors such as YY1, Snail, and Bcl-XL and what is the direct involvement of each of these factors in sensitization? The findings reported herein support the above hypothesis and establish a novel mechanism of galiximab-induced cell signaling resulting in the inhibition of the NF-κB/Snail/YY1/Bcl-XL dysregulated resistant circuit that controls the resistance in B-NHL and leading to sensitization of B-NHL cells to drug-induced apoptosis.

Materials and Methods

Cell lines and reagents

The human B-NHL cell lines Raji, Ramos, DHL4, and Daudi were purchased from the American Type Culture Collection. The AIDS-related lymphoma B cell line 2F7 was kindly provided by Dr. O. Martinez-Maza, University of California, Los Angeles, CA. The cell lines were cultured as described previously (27). All cells used in this study were within 15 passages after resuscitation. The cells were check routinely by morphology and tested for Mycoplasma contamination with CELLshipper Mycoplasma Detection Kit (Bionique Testing Laboratories). Galiximab was obtained from BiogenIdec, Inc. Soluble recombinant human TRAIL was purchased from Pepro-Tech Inc. The cis-Diamminedichloroplatinum(II) (CDDP) was purchased from Sigma and was diluted in dimethyl sulfoxide. The NF-κB inhibitor DHMEQ was provided by Dr. K. Umezawa (Keio University, Yokohama, Japan) and diluted in dimethyl sulfoxide (34). The phycoerythrin (PE)-labeled anti-CD80 antibody and the fluorescein isothiocyanate (FITC)-labeled antiactive caspase-3 antibodies, as well as the corresponding IgG1 isotype controls were obtained from BD Pharmingen. The Bcl-2 family inhibitor 2MAM-A3 was purchased from BIOMOL. The following antibodies were obtained from Santa Cruz Biotechnology: Bcl-XL, Bcl-2, p50, p65, phospho-p65 Ser-536, IκBα, phospho-IκBα Ser-32, IKK, phosphor-IKK-α/β Ser-176, AKT, phospho-AKT Thr-308, YY1, and Snail.

Viability assay

Cell viability was assessed by either the trypan blue dye exclusion assay by microscopy or by the 2,3—bis(2-methoxy-4-nitro-S-sulfophenynl)H-tetrazolium-5 carboxanilide inner salt (XTT) dye absorbance according to the manufacturer's instruction (Roche Diagnostic GmbH) and as previously described (35). The viability of the untreated cells was set at 100%. Furthermore, total cell recovery was also recorded. Each experimental condition was conducted in triplicate and the SD was calculated.

Western blot analysis for protein expression

One million of Raji cells per milliliter were incubated with or without the indicated concentrations of galiximab at 37ºC for 18 hours. Western blot analysis was conducted as previously described (35).

Electrophoretic mobility shift assay

Raji cells (106) were incubated with or without the indicated concentrations of galiximab at 37ºC for 18 hours. Ten microliters of nuclear proteins was mixed with the biotin probe for analysis of the transcription factors NF-κB, Snail, and YY1, using the EMSA kits from Panomics, following the manufacturer's instructions and as previously described (35).

Surface expression of CD80

Cells were stained with the PE-conjugated specific anti-CD80 for 1 hour at 4°C according to the manufacturer's instruction. Analysis was conducted using Flow Epics XL-MCL (Coulter) equipment. The mean fluorescence intensity was recorded using the System II Software.

Apoptosis determination

Apoptosis was assessed in tumor cells as previously described for activated caspase-3 by flow cytometry (35). In addition, we also used the Annexin-V method to corroborate the caspase-3 method.

Transfection with short interfering RNA

Transfection was conducted using the Lipofectamine transfection reagent (Invitrogen Life Technology). Scrambled RNA, Snail, and YY1 short interfering RNAs (siRNA) were obtained from Santa Cruz Biotechnology. Raji cells were cultured at a density of 2.5 × 105/mL in RPMI-1640 devoid of antibiotics for 24 hours. Cells were then transfected with 50 nmol/L siRNA in a final volume of 100 μL of medium in the presence of 10 μL of Lipofectamine 2000 in Opti-MEM. To determine YY1 or Snail siRNA-induced sensitization to CDDP- or to TRAIL-induced apoptosis, 48 hours following treatments, untransfected cells and cells transfected with scrambled siRNA, YY1 siRNA, or Snail were treated with CDDP or TRAIL for 24 hours and apoptosis was measured by FITC-labeled antiactive caspase-3 antibody using flow cytometry.

Isobologram analysis for synergy determination

The isobologram analysis was used to evaluate the effect of the galiximab/CDDP combination as described (36).

Statistical analysis

All results were expressed as the mean ± SD of data obtained from 3 triplicate independent and separate experiments. The statistical significance of differences between group means was determined using one-way ANOVA to compare variance. Significant differences were considered for probabilities <5% (P < 0.05).

Results

Galiximab-mediated inhibition of cell proliferation and sensitization of B-NHL cells to apoptosis by CDDP and TRAIL

Several B-NHL cell lines were first examined for surface CD80 expression by flow cytometry. There was differential CD80 expression ranging from low (Ramos, DHL4) to high (Raji, Daudi, 2F7) as shown in the histogram in Fig. 1A. The mean fluorescence intensity (MFI) for all the cell lines is shown in a table format depicted in Fig. 1A.

Galiximab inhibits cell proliferation of B-NHL cell lines and sensitizes B-NHL cells to apoptosis by CDDP and TRAIL. A, surface expression of CD80 on B-NHL cell lines. The surface expression of CD80 was analyzed by flow cytometry. A, representative histogram is shown for the various B-NHL cell lines and an isotype control is shown. In addition, the mean fluorescence intensity (MFI) is represented in the table. B, sensitization to apoptosis. B-NHL cell lines were treated with galiximab (20 μg/mL) for 18 hours and followed by treatment with CDDP (5 μg/mL) or TRAIL (5 ng/mL) for an additional 18 hours and apoptosis was determined by activation of caspase-3 (left) as described. *, P < 0.05; **, P < 0.01. Apoptosis was also determined by Annexin-V as described (right). C, galiximab-induced inhibition of Raji cell viability and cell recovery. The B-NHL cell line Raji was treated with various concentrations of galiximab and incubated for different time periods (6–24 hours), and cell viability was determined by trypan blue dye exclusion and total cell recovery was recorded. Raji cells that were not treated with galiximab represented 100% viability. The data represent the mean ± SD from 3 independent experiments *, P < 0.05. D, galiximab sensitizes resistant B-NHL cell lines to apoptosis by CDDP and TRAIL. Sensitization of B-NHL Raji cells by galiximab to apoptosis by CDDP or TRAIL is synergistic. Raji cells were treated with various concentrations of galiximab (10–100 μg/mL) for 18 hours and then treated with either CDDP (5, 10, and 20 μg/mL; left) or only one concentration of galiximab (20 μg/mL) and various concentrations of TRAIL (2.5, 5, and 10 ng/mL; right) for an additional 18 hours and apoptosis was determined. The data represent the mean ± SD from 3 independent experiments. *, P < 0.05; **, P < 0.01. In addition, the data were analyzed for synergy by isobologram analysis as described in Materials and Methods. The isobologram is represented left of D. AAD, aminoactinomycin D.

The resistant B-NHL cell lines were tested for galiximab-mediated sensitization to apoptosis by measuring the activation of caspase-3 by flow cytometry following the addition of suboptimal concentrations of CDDP and TRAIL. As shown in Fig. 1B (left), single-cell treatment with galiximab (20 μg/mL) had no significant apoptotic effect on the cell lines; however, with the combination of galiximab and drug, there was significant sensitization to both CDDP and TRAIL apoptosis in all of the B-NHL cell lines tested. The single-cell treatment with CDDP or TRAIL had no significant apoptosis. The level of apoptosis achieved was different for each cell line tested. 2F7, Raji, and Daudi cells were significantly sensitized by both CDDP and TRAIL. Ramos, however, was not sensitized to CDDP, although it was sensitized to TRAIL. The DHL4 cell line was not sensitized to CDDP in combination with galiximab treatment but was sensitized to TRAIL (data no shown). The apoptotic effect assessed by the activation of caspase-3 was corroborated by analysis of apoptosis using Annexin-V as shown in Fig. 1B (right).

We have chosen Raji cells as a model for further analysis of the underlying mechanism of galiximab-mediated sensitization. Treatment of Raji cells with various concentrations of galiximab resulted in a concentration-dependent inhibition of cell survival. Concentrations of galiximab ≥25 μg/mL resulted in a decrease of viability and a plateau (75%–80% viability) was reached at concentrations ≥25 μg/mL. In addition, there was inhibition of cell proliferation by galiximab concentrations of ≥10 μg/mL as determined by total cell recovery (Fig. 1C). The effects of various galiximab concentrations as well as different concentrations of CDDP and TRAIL were examined for sensitization to apoptosis. Raji cells were pretreated with various concentrations of galiximab (10–100 μg/mL) for 18 hours and then treated for an additional 24 hours with CDDP (5–20 μg/mL) or TRAIL and apoptosis was determined. The findings show that there was significant sensitization by the combination treatment and the level of apoptosis was a function of the concentrations used for both galiximab and CDDP (Fig. 1D, left). The combination treatment was synergistic as determined by isobologram analysis. Likewise, the combination of galiximab and TRAIL resulted in apoptosis and the level of apoptosis was a function of the increased concentration of TRAIL (Fig. 1D, right).

Mechanism by which galiximab sensitizes Raji cells to apoptosis by CDDP and TRAIL

Inhibition of NF-κB activity.

We examined whether galiximab, due to its antiproliferative activity, inhibited the constitutively activated NF-κB pathway in Raji cells. Raji cells were treated with several concentrations of galiximab (25, 50, and 100 μg/mL) for 18 hours, and cell lysates were prepared for analysis for the expression of several gene products of the NF-κB pathway. Western blot and densitometric analyses revealed that following galiximab treatment, there was some inhibition of p50 but there were significant inhibition of p65, phospho-p65, and both phospho- and total IκBα expression levels in a concentration-dependent manner (Fig. 2A, left) and quantified by densitometric analysis (Fig. 2A, right). The inhibition of NF-κB DNA-binding activity by galiximab and by the specific NF-κB inhibitor DHMEQ was corroborated by electrophoretic mobility shift assay (EMSA; Fig. 2B). To show the involvement of NF-κB inhibition by galiximab in the reversal of resistance, the tumor cells were treated with the specific NF-κB inhibitor, DHMEQ, and tested for sensitivity to apoptosis induced by CDDP and TRAIL. Treatment with DHMEQ sensitized the tumor cells to apoptosis by CDDP and TRAIL and, thus, mimicking the treatment with galiximab (Fig. 2C). The effect of galiximab treatment on the AKT pathway was examined by Western blot analysis. Galiximab inhibited phospho-AKT and phospho-IKKα/β but not the unphosphorylated forms as shown by Western blot analysis and densitometry (Fig. 2D).

Galiximab inhibits NF-κB activity in Raji cells and the role of NF-κB inhibition in the sensitization of Raji to apoptosis by CDDP and TRAIL. Raji cells were treated with different concentrations of galiximab (25, 50, and 100 μg/mL) for 18 hours, and aliquots were used to prepare both nuclear and total cell lysates as described in Materials and Methods. A, Western blot analysis for NF-κB expression. Total cell lysates were tested for various gene products of the NF-κB pathway. β-Actin was used as a loading control. Densitometric analysis is also shown and intensity of the bands was normalized to β-actin bands. B, inhibition of NF-κB DNA-binding activity by galiximab. Raji cells were treated with galiximab (25 μg/mL). Nuclear lysates were tested for NF-κB DNA-binding activity by EMSA as described. The NF-κB inhibitor DHMEQ (10 μg/mL) was used as a positive control and cold probes as competitors. For the supershift assay, the nuclear proteins were incubated with anti-p65 antibody overnight at 4ºC before the analysis by EMSA. C, galiximab-induced inhibition of NF-κB in the sensitization to apoptosis by CDDP and TRAIL. Raji cells were treated with galiximab (20 μg/mL) for 18 hours or with the NF-κB inhibitor DHMEQ (10 μg/mL) for 18 hours and the cells were subsequently treated with either CDDP (5 μg/mL) or TRAIL (5 μg/mL) for an additional 24 hours and apoptosis was determined. The data represent the mean ± SD from 3 independent experiments. *, P < 0.01. D, inhibition of the AKT pathway by galiximab. Total cell lysates were tested for various gene products of the AKT pathway. β-Actin was used as a loading control. Densitometric analysis is also shown.

Inhibition of the resistant factors YY1 and Snail by galiximab; inhibition of the expression and activity of both YY1 and Snail by galiximab.

We and others have reported that several antiapoptotic gene products regulated by NF-κB participate in the acquisition of tumor cell resistance to apoptotic stimuli (37–39). For instance, the transcriptional repressor YY1 was shown to inhibit the response to TRAIL apoptosis via the TRAIL receptor DR5 (40). Furthermore, the transcription repressor Snail was also reported to participate in the antiapoptotic activity of tumor cells (41). NF-κB regulates the transcription of both Snail (31) and YY1 (32) and, in addition, Snail transcription is also regulated by YY1 (42). Therefore, we examined whether galiximab-induced inhibition of NF-κB also inhibited the expression and activity of both YY1 and Snail. Raji cells were treated with various concentrations of galiximab (25, 50, and 100 μg/mL) for 18 hours and both nuclear and total cell lysates were prepared. Western blot analysis revealed that galiximab significantly inhibited both YY1 and Snail expressions in a concentration-dependent manner and quantified by densitometric analyses (Fig. 3A). The inhibition of the DNA-binding activities of YY1 and Snail by galiximab was determined by EMSA (Fig. 3B).

Galiximab inhibits the expression and the activity of the transcription factors YY1 and Snail in Raji cells. A, inhibition of YY1 and Snail by galiximab. Raji cells were treated with various concentrations of galiximab (25, 50, and 100 μg/mL) for 18 hours and total cell lysates were prepared for Western blot analysis. β-Actin was used as a loading control. The Western blots analyses were also analyzed by densitometry and is shown below the Western blot analysis figure. B, galiximab inhibits the DNA-binding activity of YY1 and Snail. Raji cells were treated with galiximab (25 μg/mL) for 18 hours and nuclear lysates were tested for DNA-binding activities for YY1 and Snail as described in Materials and Methods. The specificity of DNA-binding activity was determined by the use of a corresponding competitive cold probe and in the absence of nuclear extracts in the assay.

The direct role each of YY1- and Snail-induced inhibition by galiximab in the sensitization of Raji tumor cells to apoptosis by CDDP and TRAIL.

Raji cells were treated with YY1 siRNA or control siRNA and Western blot analysis was conducted. Treatment with YY1 siRNA inhibited YY1, Snail, and p-p65. Treatment with Snail siRNA inhibited Snail, p-p65, and p-AKT (Fig. 4A). The siRNA-transfected cells were then treated with CDDP or TRAIL and examined for apoptosis. Whereas treatment with control siRNA did not reverse resistance, treatment with YY1 siRNA significantly sensitized the cells to apoptosis by both CDDP and TRAIL (Fig. 4B, top). Treatment of Raji cells with Snail siRNA, unlike control siRNA, significantly sensitized the tumor cells to apoptosis by both CDDP and TRAIL (Fig. 4B, bottom).

The involvement of YY1- and Snail-induced inhibition by galiximab and downstream inhibition of Bcl-XL in the sensitization of Raji cells to apoptosis by both CDDP and TRAIL. A, transfection with YY1 siRNA and Snail siRNA and sensitization. Raji cells were transfected with YY1 siRNA, Snail siRNA, or control siRNA. Treatment with YY1 siRNA but not control siRNA inhibited the expression of YY1, Snail, and phospho-p65. Treatment with Snail siRNA inhibited the expression of Snail, phospho-p65, and phospho-AKT. β-Actin was used as a loading control. B, the direct role of YY1 and Snail in sensitization. Sensitization of Raji cells to TRAIL- and CDDP-induced apoptosis following treatment with YY1 siRNA (top) or with Snail siRNA (bottom). *, P < 0.05. C, inhibition of Bcl-2 and Bcl-XL by galiximab. Raji cells were treated with various concentrations of galiximab (25, 50, and 100 μg/mL) and cell lysates were tested for Bcl-2 and Bcl-XL expression. β-Actin was used as loading control. D, inhibition of Bcl-XL expression by knocking down YY1 and/or Snail. Raji cells were transfected with either YY1 siRNA, Snail siRNA, or control siRNA and lysates were examined for Bcl-XL and Bcl-2 expression by Western blot analysis. β-Actin was used as a loading control. E, the role of Bcl-2 inhibition in the sensitization to CDDP and TRAIL. Raji cells were treated with the pan-Bcl-2 family inhibitor 2MAM-A3 for 18 hours and then treated with either CDDP or TRAIL for an additional 18 hours and apoptosis was determined. *, P < 0.05.

Mechanism of YY1- and Snail-induced inhibition by galiximab in the reversal of resistance: the role of Bcl-XL–induced inhibition by galiximab, YY1 and Snail in the sensitization to apoptosis by CCDP and TRAIL.

The antiapoptotic Bcl-2/Bcl-XL factors in tumor cell resistance are well established (26, 27). Both Bcl-2 and Bcl-XL expressions were inhibited by galiximab in a concentration-dependent manner (Fig. 4C). We examined whether the sensitization was mediated by the inhibition of Bcl-2/Bcl-XL gene products by YY1 and Snail in Raji cells. Treatment with YY1 siRNA, and not with control siRNA, resulted in the specific inhibition of both Bcl-2 and Bcl-XL (Fig. 4D). However, treatment with Snail siRNA inhibited significantly Bcl-XL expression and modestly Bcl-2 (Fig. 4D). These findings showed that both Snail and YY1 participate in the regulation of Bcl-XL expression and their role in sensitization was tested following treatment of Raji cells with the pan-Bcl-2 family inhibitor 2MAM-A3. Such treatment reversed the resistance of Raji cells to apoptosis by both CDDP and TRAIL (Fig. 4E).

Altogether, the above findings show that galiximab inhibits NF-κB activity and downstream both YY1 and Snail. Individually, the inhibitions of either one of those factors reverse the resistance. In addition, those factors have in common the regulation of the antiapoptotic gene product Bcl-XL whose inhibition reverses the resistance.

Discussion

Galiximab (anti-CD80 monoclonal antibody) is currently being investigated as a novel therapeutic against B-NHL and is currently in phase III clinical trials (43). A randomized, double-blind study of galiximab in combination with rituximab was compared with rituximab in combination with placebo for the treatment of subjects with relapsed or refractory, follicular NHL. The results show that the addition of galiximab to rituximab reduced the hazard for disease progression or death by 26% compared with the rituximab + placebo group. Galiximab has shown some clinical benefits; however, the mechanism by which galiximab mediates its antitumor effects is not clear. The present in vitro findings show that galiximab exerts an antiproliferative response on Burkitt's B-NHL cell lines and also sensitizes the resistant tumor cells to apoptosis by both chemotherapeutic and immunotherapeutic drugs. Galiximab signals the cells via the CD80 receptor and inhibits intracellular survival/antiapoptotic pathways such as the NF-κB and AKT pathways and their targets, namely, the antiapoptotic transcription factors YY1 and Snail and each leading to inhibition of the antiapoptotic gene product Bcl-XL. Individually, inhibition by galiximab of NF-κB, YY1, Snail, or Bcl-XL in B-NHL cells results in the sensitization to both CDDP- and TRAIL-mediated apoptosis. These findings establish the presence of a dysregulated NF-κB/Snail/YY1/Bcl-XL resistant circuit in B-NHL cells and its inhibition by galiximab leads to the reversal of drug resistance.

Galiximab inhibited the expression of phospho-p65, a member of the NF-κB pathway, as well as NF-κB DNA-binding activity. It is not clear that how galiximab inhibits NF-κB activity. CD80 is a transmembrane immune costimulatory glycoprotein involved in the regulation of T-cell activation (44). CD80 also serves as a receptor that transduces distinct signals to the cells expressing CD80 upon engagement by CD28 (45). It is possible that it translocates the CD80 receptor into lipid rafts and inhibits Src kinases upstream of NF-κB as we and others have previously shown following treatment of B-NHL cells with rituximab (26, 27). The involvement of galiximab-induced inhibition of NF-κB in sensitization was corroborated by the use of the NF-κB–specific inhibitor DHMEQ which mimicked galiximab in sensitizing Raji cells to apoptosis by both CDDP and TRAIL.

We report here a mechanism by which galiximab-mediated inhibition of NF-κB and AKT resulted in the reversal of resistance. We show the presence of a dysregulated NF-κB/YY1/Snail/Bcl-XL resistant circuit and whose inhibition by galiximab reverses resistance. Each of the gene products individually participated in the reversal of resistance following their inhibition by galiximab. Galiximab inhibited the expression of the transcription factor YY1 that is regulated, in part, by NF-κB (40). We show here the direct role of YY1 inhibition by galiximab in the reversal of resistance using YY1 siRNA in agreement with our previous findings (40, 46). The involvement of YY1 in the regulation of the apoptotic pathway is shown here by its ability to regulate the expression of Bcl-XL and Bcl-2 as analysis of the promoters revealed the presence of putative YY1-binding sites.

Galiximab inhibited Snail expression and Snail is involved in the regulation of tumor cell response to CDDP and TRAIL as shown here in cells transfected with Snail siRNA. Like YY1, treatment with Snail siRNA also inhibited Bcl-XL expression. The mechanism by which Snail inhibits Bcl-XL is not clear. Because Snail is primarily a repressor and there are no putative binding sites of Snail on the Bcl-XL promoter, we envisaged that Snail inhibits the regulation of Bcl-XL indirectly (47). Studies through activation of the mitogen-activated protein kinase (MAPK) and PI3K/AKT pathways showed that Snail-expressing cells show hyperactivation of MAPK and PI3K/AKT activities. Both pathways can modulate the upregulation of Bcl-XL expression (48). We show here that the treatment with Snail siRNA inhibited phospho-AKT. Also, Snail negatively regulates Wnt, which encodes a secreted Wnt family of proteins that negatively regulate NF-κB activity and downstream Bcl-XL (49). The involvement of galiximab-induced inhibition of Snail and YY1 and their regulation of Bcl-2 and Bcl-XL leading to sensitization was corroborated in experiments showing that the pan-Bcl-2 inhibitor 2MAM-A3 sensitized the tumor cells to CDDP and TRAIL apoptosis.

Our findings in this report are schematically diagrammed in Fig. 5 and can be summarized as follows: the constitutively activated NF-κB and AKT pathways in Raji cells lead to the expression of YY1 and Snail and to the overexpression of antiapoptotic gene products such as Bcl-2 and Bcl-XL. Treatment with galiximab inhibits NF-κB and AKT activities and downstream the expression of YY1 and Snail, leading to inhibition of Bcl-2 and Bcl-XL and reversal of resistance. The present findings support the existence of a dysregulated NF-κB/YY1/Snail/Bcl-2/Bcl-XL resistance circuit as each of the gene products in this circuit is inhibited by galiximab and each directly regulates tumor cell resistance. These findings offer a potential therapeutic approach using the combination of galiximab and either subtoxic chemoimmunotherapeutic drugs or specific inhibitors of the resistant factors in the circuit in the treatment of drug-resistant CD80-expressing hematologic malignancies.

Schematic diagram representing the mechanism by which galiximab sensitizes B-NHL cells to both chemo- and immunocytotoxic drugs. Raji cells exhibit constitutively activated NF-κB and AKT activities and resulting downstream in the expression of Snail, YY1, and Bcl-2/Bcl-XL. These resistant gene products regulate the resistance to apoptotic stimuli. However, treatment with galiximab inhibits NF-κB and AKT activities and downstream in the inhibition of Snail, YY1, and Bcl-2/Bcl-XL expressions. Each of these gene products, namely, Snail, YY1, or Bcl-2/Bcl-XL, inhibited by galiximab result in the reversal of resistance and sensitization to both CDDP and TRAIL apoptosis. Hence, the tumor cells exhibit a constitutively dysregulated NF-κB/Snail/YY1/Bcl-2-Bcl-XL resistant circuit whose inhibition by galiximab results in the reversal of resistance when used in combination with cytotoxic drugs.

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Acknowledgments

The authors thank the assistance of Kerry Choy, Daphne Liang, and Melissa Cao in preparation of the manuscript and Dr. Kazuo Umezawa for the NF-κB inhibitor. They also thank Programa de Posgrado; Doctorado en Ciencias Biomédicas, Facultad de Medicina UNAM, and Dr. Otoniel Martinez-Maza for valuable input and support.